The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
A number of assays based on bioaffinity binding reactions or enzymatically catalyzed reactions have been developed to analyze biologically important compounds from or their activity or their biological effect or its modulation induced by various biological samples, samples in environmental studies, industrial processes and compound libraries. Some of these assays rely on specific bioaffinity recognition reactions, where e.g. natural biological binding components, artificially produced binding compounds or moulded plastic imprints (molecular imprinting) are used as recognition elements to form the specific binding assay. Other assays rely on activity or modulation of the activity of compounds present in sample or added into reaction (e.g. biologically active enzymes, chemical compounds with activity on biological molecules, enzyme substrates, enzyme activators, enzyme inhibitors, enzyme modulating compounds) and so on. Such assays generally rely on a label or a combination of multiple labels generating signals to e.g. quantitate the formed complexes after recognition and binding reactions. In heterogeneous assays a separation step (separations like precipitation and centrifugation, filtration, affinity collection to e.g. plastic surfaces such as coated assay tubes, slides or microparticles, solvent extraction, gel filtration, or other chromatographic systems, and so on) is generally required before e.g. the free or bound fraction of the label signal can be measured. In homogeneous assays the signal of the label or labels is modulated or formed due to binding reaction or enzymatic activity or other measured effect, and no separation step is needed before measurement of the label signal. Both in heterogeneous and homogeneous assays the measurement of the label signal from free or bound fraction of the label generally enables the calculation of the analyte or activity in the sample directly or indirectly, generally through use of a set of standards to which unknown samples are compared. Various binding assay methods have been reviewed in Principles and Practice of Immunoassay, 2nd ed., C. P. Price and D. J. Newman, eds., Palgrave Macmillan, Hampshire, UK, 2001; and The Immunoassay Handbook, 2nd ed. David Wild, ed., Nature Publishing Group, New York, N.Y., 2001.
Development of simple, sensitive, and quantitative, preferably homogeneous and multiplexed nucleic acid hybridization assays has been an important objective in evolution of fluorescent labels and detection techniques. Homogeneous methods have received much attention, because they eliminate the need for cumbersome steps of separation of bound and free label, and significantly simplify construction of an instrument required to perform an assay automatically. Further, homogeneous methods are required for e.g. techniques involving real-time monitoring of nucleic acid amplification reactions [Higuchi, R., et al. (1992) Biotechnology 10: 413-417; Higuchi, R., et al. (1993) Biotechnology 11:1026-1030]. Currently available label technologies suitable for homogeneous, non-separative monitoring of nucleic acid hybridization still suffer from interference of sample matrices, the technologies cannot be universally employed, e.g. are not suitable for 5′ nuclease assays [U.S. Pat. No. 5,210,015], or they simply do not enable detection sensitive enough to be performed using a rapid read-out required, or the instrumentation required for detection is too complex or expensive to be feasibly constructed or miniaturized.
Homogeneous detection techniques based on photoluminescence have received much attention, since several types of physical and chemical interactions can be employed to modulate the emission of photoluminescent labels due to formation of specific biomolecular complexes. The commonly employed methods are based on polarization of the emitted light or non-radiative energy-transfer (resonance energy transfer) between two photoluminescent compounds (donor and acceptor) or between a photoluminescent and a non-luminescent compound (donor and quencher) [Hemmilä I, Clin Chem 1985; 31:359-370].
Resonance Energy Transfer
Förster resonance energy transfer (FRET) is a strongly distance dependent (to inverse sixth power) non-radiative energy transfer mechanism between two properly chosen fluorescent molecules present in close proximity [Förster, T (1948) Ann Physik 2: 55-75]. Resonance energy transfer (RET) occurs at practical efficiency when a donor and an acceptor fluorophore are within Förster radius (typical values 4-7 nm) and the donor emission spectrum and the acceptor absorption spectrum overlap. The RET is typically monitored either by measuring a decrease of donor emission or an increase of acceptor emission intensity (known as sensitized acceptor emission) [Selvin, P R (1995) Biochem Spectroscopy 246: 300-334] resulting from proximity of donor and acceptor. In case of non-fluorescent acceptor (known as quencher) a change of donor emission intensity is monitored.
Although FRET is a widely employed and an essential technique in many applications, it has severe performance limitations [Hemmilä, I (1985) Clin Chem 31: 359-370.] and, in practice, the RET probes fail to comply with the strict requirements of true proximity probes. Proximity probing is a technique capable of detecting the nearness of the two proximity probes and is used for specific, sensitive and rapid detection of various biomolecules. A proximity probe consists typically of a binding moiety (recognition element) or other recognition site (with specific affinity for the target molecule i.e. analyte) and the target molecule is able to direct binding of the two similar or different proximity probes into adjacent positions. The proximity between the probes is thus provided when two probes bind e.g. to their respective binding sites on a target molecule. Characteristics to the true proximity probes is that they do not generate any significant signal (i.e. are not detectable) when the probe pair is not in immediate proximity directed by the target molecule, but the probe pair is switched to a detectable state due to specific recognition events in presence of target molecule. Proximity probing using monovalent proximity probes, performed in solution with no washing steps, has been described in WO 01/61037; Schallmeiner et al. (2006) Nature Methods 4: 135-137 and WO/2003/044231.
The conventional FRET-based assays are susceptible to i) direct excitation of the acceptor (the acceptor is weakly excited at the same wavelength where the donor is excited), ii) crosstalk of donor emission (the donor has some emission at the same wavelength where the acceptor emission is measured), iii) radiative energy transfer (less distance dependent; to inverse second power) through absorption of donor emission (photons) by acceptor fluorophores not necessarily in proximity, and iv) scattered excitation light and autofluorescence (from sample, other assay components, plastics and detection instrument itself) generating background signal. Thus, conventional fluorophores and RET probes do not provide the specificity in signal generation required for the proximity probe binding-principle. Further, it is difficult to measure more than two parameters simultaneously in a multiparametric FRET-based assay due to wide spectral coverage of an individual donor-acceptor pair.
Time-Resolved Fluorometry
Detection sensitivity of conventional fluorescence based techniques is limited by autofluorescence, scattered excitation light and absorbance of biological sample matrices, and in acceptor-based resonance energy transfer based assays also by crosstalk of the emission of the donor at acceptor-specific emission wavelength and direct excitation of the acceptor at the donor-specific excitation wavelength. Many compounds and proteins present in biological fluids or serum are naturally fluorescent, and the use of conventional fluorophores leads to serious limitations of sensitivity [Soini E and Hemmilä I (1979) Clin Chem 25: 353-361; Wu P and Brand L (1994) Anal Biochem 218:1-13]. Another major problem when using homogeneous fluorescence techniques based on intensity measurements is the inner filter effect and the variability of the optical properties of a sample. Sample dilution has been used to correct this drawback, but always at the expense of analytical sensitivity. Feasibility of fluorescence resonance energy transfer in assay applications was significantly improved when fluorescent lanthanide cryptates and chelates with long-lifetime emission and large Stokes' shift were employed as donors in the 1990's [Mathis G (1993) Clin Chem 39:1953-1959; Wu P and Brand L (1994) Anal Biochem 218, 1-13; Selvin P R et al. (1994) Proc Natl Acad Sci USA 91:10024-10028; Stenroos K et al. (1998) Cytokine 10:495-499; WO 98/15830; U.S. Pat. No. 5,998,146; WO 87/07955; Blomberg, K et al. (1999) Clin Chem 45:855-61].
Lanthanide chelates and cryptates, due to their enhanced detectability compared to traditional organic fluorophores, are nowadays widely used in the analysis of various biological molecules. Luminescent chelates of lanthanides (rare earths, e.g. trivalent europium, terbium, samarium and dysprosium) are an exceptional group of photoluminescent compounds [Bünzli, J C G and Piguet, C (2005) Chem Soc Rev 34: 1048-1077]. The lanthanide ions themselves have very low absorption and, in addition, the excited state of the lanthanide is efficiently quenched by coordinated water molecules. Thus, the only practical solution to their excitation is to use a coordinating ligand comprising a light harvesting moiety, such as an organic antenna chromophore in the intrinsically luminescent lanthanide(III) chelate. In practice, the photoluminescence efficiency (product of absorption coefficient and quantum yield) of the lanthanide ion chelated to an efficient antenna ligand, displacing all the coordinating water molecules, can be readily enhanced up to 100 000-fold compared to a bare ion. Further, the distinct emission bands characteristic to lanthanide ion enable simultaneous measurement of up to four different lanthanides with minimal spectral crosstalk. The luminescence properties of lanthanides enable also efficient separation of the background noise from biological material and thus increase of the sensitivity of the assay [J. Yuan, G. Wang (2005) J Fluoresc. 15, 559].
Lanthanide ions complexed to a suitable chelate (e.g. aminopolycarboxylic acid) containing organic light harvesting antenna moiety or chromophore possess unusual fluorescence characteristics compared to conventional fluorophores: large Stokes shift (150-300 nm), narrow and distinct emission bands characteristic to lanthanide ions, and long luminescence lifetime (up to 2000 microseconds). The exceptional fluorescence lifetime enables efficient background separation by selection of such a temporal gate (typically hundreds of microseconds) that detection is performed only when the background fluorescence (short living) has decayed away, while the lanthanide luminescence is still reasonable intense. Moreover, the large Stokes shift and the narrow emission bands enable efficient wavelength filtering to spectrally select the lanthanide luminescence, resulting in highly sensitive reporter technology (equal performance to enzyme amplified chemiluminescence) and possibility for multiparametric measurement. The technology utilizes a dedicated detection method known as (microsecond) time-resolved fluorometry [Soini E and Kojola H (1983) Clin Chem 29: 65-68]. The long-lifetime fluorescence of luminescent lanthanide chelates is typically excited at ultraviolet or blue visible light [Yang C, et al. (2004) Angew Chem Intl Ed 43: 5010-5013] and the emission is detected at green and red visible wavelengths. In case of erbium, neodymium and ytterbium the excitation can be at visible wavelenghts and the emission at visible or at infrared wavelengths [Werts, M. H. V., et al. (1997). Chem Phys Lett 276: 196-201]. Also platinum (III) and palladium (III) should be noted to have similar spectral and temporal properties when complexed to phorphyrins [de Haas, R. R., et al. (1999) J Histochem Cytochem 47: 183-196].
The excitation mechanism of lanthanide(III) chelates, where an organic light harvesting antenna is used to excite the emissive lanthanide(III) ion via energy-transfer, is exceptional among fluorescent reporters [Hemmilä, I. and Laitala, V. (2005) J Fluoresc 15: 529-542]. Luminescent lanthanide(III) chelates comprise a reactive group, light-harvesting antenna and chelating groups, which chelate the lanthanide(III) ion through coordination bonds. The organic light harvesting chromophore is first excited from ground singlet state (S0) to first singlet state (S1) by light absorption, and the chromophore undergoes transition to triplet state (T1) by intersystem crossing (ISC). The triplet state of the antenna chromophore can transfer the excitation energy to appropriate 4f energy level of the lanthanide(III) ion. Thereafter, the lanthanide ion produces characteristic f-f transition luminescence with distinct emission bands and with a long luminescence lifetime due to forbidden transition.
The development of a stable lanthanide chelate structure containing an efficient light-harvesting antenna originally turned out difficult. The problem was circumvented in heterogeneous assays by labelling the biomolecular binder with an ion carrier chelate and using a separate chelating solution (with low pH) to dissociate the ion from the carrier chelate to form a new highly fluorescent lanthanide complex. The ion carrier chelate used for labelling contain, in addition to the lanthanide ion and carrier chelate, a reactive group for covalent coupling.
The chelate complexes of metals (coordination compounds) are formed through binding of ligand (or chelating molecule) to metal ion through coordinated groups. The total number of points of attachment of the ligand to the central metal ion is termed the coordination number. The ligands can be characterized for points of attachment, listing them as monodentate, bidentate, etc., where the concept of teeth (dent) reflects the number of atoms bonded to the metal centre in the chelate. The chelate (or chelate complex) is a compound that comprises at least a single ligand, which has at least two teeth (called bidentate), and at least one metal ion bound by the ligand. The stability of chelate complexes in solution is described by the magnitude of stability (or formation) constant for association of the metal (cations) to ligands (neutral or anionic). The larger the stability (or formation) constant, the higher proportion of the metal is complexed in presence of the ligand. For binding of multiple ligands stepwise stability constants can be defined and the stability constant is then the product of stepwise stability constants. Since the stability constants can vary with tens of magnitudes, the value is typically expressed as logarithm (log 10). Multidentate ligands form stronger metal ion complexes than monodentate ligands. Typically the stability constant increases with number of coordination dentates of the ligand, but in addition the structure of the ligand is important. Ring or cyclic structures reducing the freedom of conformations of the binding ligand often also result in higher stability constants. Determination of stability constants for europium(III) complexes is described e.g. by Wu, S L and Horrocks, W D (1997) Journal of the Chemical Society-Dalton Transactions 1497-1502. Typically neighbouring lanthanides (e.g. Eu(III) and Gd(III)) in the periodic table have very similar stability constants with the same ligand.
The stability (or formation) constant describes the maximum stability of the lanthanide chelate at alkaline conditions, where the ligand is fully deprotonated and the protons do not significantly compete with binding to metal ion. The conditional stability constants (known also as effective formation constant), taking into account the pH and prototation of the ligand, are more appropriate to describe the actual stability of the complex at e.g. physiological pH and conditions typically prevailing in bioassays. Description of the terms “Determination of conditional stability constants for europium(III) complexes is described e.g. by Siaugue, J. M. et al. (2003) J Photochem Photobiol A: Chem 156: 23-29.
Examples of both stability constants and conditional stability constants at physiological pH as well as kinetic stability data are described in Morcos, S. K. (2007) “Chelates and stability”, pp. 155-160 in Medical Radiology, 2nd revised edition by Thomsen, H. S. and Webb, J. A. W, Springer, Berlin, 2009. A large collection of stability constants is compiled to the IUPAC stability constant database commercially available from Academic Software, Yorks, United Kingdom.
The technique based on an ion carrier chelate and a separate chelating solution was known as dissociation enhanced lanthanide fluoroimmunoassay assay [U.S. Pat. No. 4,565,790; Hemmilä, I et al. (1984) Anal Biochem 137: 335-343; Soini, E and Lövgren, T (1987) CRC Crit Rev Anal Chem 18: 105-154; and Siitari, H et al. (1983) Nature 301: 258-260]. The technology is widely applied in heterogeneous biomolecular binding assays and has later been improved to speed up the dissociation by utilizing an antenna ligand being able to form lanthanide complex at lower pH [WO 2003/076939, U.S. Pat. No. 7,211,440, U.S. Pat. No. 7,381,567 and EP 1 483 582]. The enhancement-based assays typically utilize moderately strong aminopolycarboxylate-based lanthanide (III) ion carrier chelates (such as derivatives of EDTA and DTPA, described in e.g. U.S. Pat. No. 4,822,594 and U.S. Pat. No. 6,190,923) as labelling reagents and beta-diketone-based antenna ligands in enhancement solution to create luminescence. Also ion carrier chelates and labelling reagents based on DOTA and TETA have been presented [Hemmilä, I. (1995) J. Alloys Comp 225: 480-485]. To derivatize the ligand for labelling, e.g. one carboxylic acid of DOTA can be replaced with a group for attachment to biomolecules. The stability of the lanthanide(III) ion carrier chelates used for dissociation enhancement, however, should be only moderate and the dissociation kinetics quite rapid especially at low pH, as otherwise the ion is not released fast enough for fluorescence enhancement. On the other hand, the development of very stable carrier chelates for gadolinium(III) ion has been in focus in development of contrast agents for magnetic resonance imaging [Brücher, E. (2002) Topics in Current Chemistry 221: 103-122; Morcos, S. K. (2007) “Chelates and stability”, pp. 155-160 in Medical Radiology, 2nd revised edition by Thomsen, H. S. and Webb, J. A. W, Springer, Berlin, 2009; and Woods, M. et al. (2006) Journal of Supramolecular Chemistry, Vol 2., 1-15].
Several intrinsically fluorescent lanthanide chelates have been developed [Alpha, B et al. (1987) Angew Chem Int Ed Engl 26: 1266-1267; H. Takalo et al. Bioconjugate Chem. 1994, 5, 278; Takalo, H et al. (1997) Helv Chim Acta 80: 372-387; von Lode, P et al. (2003) Anal Chem 75: 3193-3201; Beeby, A. (2000) J. Chem. Soc., Perkin Trans. 2, 1281-1283; Hakala, H. et al. (2002) Inorg Chem Comm 5: 1059-1062; Li, M. and Selvin, P. R. (1995) JACS 117: 8132-8138; and WO 2005/021538]. These stable, luminescent lanthanide complexes include both cryptates and highly luminescent chelates (mainly aminopolycarboxylic based chelating structures) for several lanthanides [europium(III), terbium(III), samarium(III) and dysprosium(III)]. The chelating ligands are designed to combine a moderately strong or strong binding of the lanthanide(III) ion and light-harvesting part to the one and same molecule and they can be used as donors in FRET based assays. In most of the chelates, the light-harvesting (energy-absorbing) and mediating part is composed of derivatized pyridine or pyridine manifold. Some antenna structures contain other heteroatomic conjugated ring structures such as pyrazole. In addition to the lanthanide ion, light-harvesting organic moiety and carrier ligand, the intrinsically luminescent lanthanide complexes used for labeling contain a reactive group for covalent conjugation.
Lanthanide luminescence yield can be enhanced by co-luminescence based enhancement utilizing additional antenna ligands and non-luminescent lanthanide ions [e.g. yttrium(III) or gadolinium(III)] to absorb excitation light and transfer the energy via triplet-triplet migration to an antenna ligand coordinated to a luminescent lanthanide ion [e.g. europium(III)], that is present either in the same self-assembled polymeric lanthanide complex or in the same micellar environment. Intermolecular energy migration greatly enhances the number of effective light harvesting antennas per luminescent lanthanide and results in enhancement of the luminescence intensity of certain luminescent lanthanide ions up to hundred-fold or even more [Xu, Y Y et al. (1991) Analyst 116: 1155-1158; Latva, M et al. (1995) J Chem Soc Perkin Trans 2 995-999].
Lanthanide-Based RET
Two novel resonance energy transfer-based methods utilizing different photoluminescent lanthanide-based reporters [Mathis, G (1993) Clin Chem 39: 1953-1959; Blomberg, K et al. (1999) Clin Chem 45: 855-861] have been introduced to largely solve the major problems associated with conventional FRET-based homogeneous assays. Both of these methods provide significant advantages compared to the conventional methods, but the specificity in signal generation is still limited by the radiative energy transfer (absorption of donor emission), especially when the labeled probes are present in high concentration (e.g. to achieve a large dynamic range, or to facilitate binding in case of weak interactions) [H. Bazin, M. et al. (2001) Spectrochim. Acta, Part A, 57]. Excess of the unbound acceptor result in slowly-decaying radiative background signal at the acceptor-specific wavelength, but also donor cross-talk at the measurement wavelength can increase the background signal unless sufficient spectral resolution is used. The utilization of non-overlapping acceptor (non-overlapping FRET) with lanthanide chelate donor [Hemmilä, I. and Laitala, V. (2005) Anal. Chem. 77:1483-1487; Laitala, V. and HemmiläI. (2005) Analytica Chimica Acta 551: 73-78] can further eliminate possible background through reabsorption of donor emission.
In case of a long-lifetime fluorescent lanthanide chelate (or cryptate) as a donor in combination with a conventional, short-lifetime fluorescent acceptor [Mathis, G (1993) Clin Chem 39: 1953-1959; Blomberg, K et al. (1999) Clin Chem 45: 855-861] the energy-transfer excited acceptor emission can be temporally resolved (with time-resolved fluorometry) from the short-lifetime, directly-excited fluorescence of the acceptor and the background fluorescence. The crosstalk of donor emission to acceptor emission wavelength is also nearly completely avoided due to narrow “line like” emission bands of donor emission. The same advantages are obtained by using upconverting (anti-Stokes photoluminescent) lanthanide-doped compounds [Heer, S et al. (2004) Adv Mater 16: 2102-2105; Kuningas, K et al. (2005) Anal Chem 77: 7348-7355] as donors in combination with a conventional, fluorescent acceptor and measuring the energy-transfer excited acceptor emission specifically at visible wavelengths under infrared excitation of the donor. The infrared illumination does not directly excite the conventional fluorescent acceptor nor generate any autofluorescence at visible wavelengths, and the narrow banded donor emission effectively eliminates the potential crosstalk.
Anti-Stokes emission of up-converting lanthanide-doped nanocrystals occurs at shorter wavelength (at visible wavelengths) than infrared excitation, providing a large anti-Stokes shift (up to over 300 nm) and efficient spectral separation of the autofluorescence and scattered excitation light (without temporal resolution) from the emission at visible wavelengths [Soukka, T. et al. (2005) J Fluorescence 15: 513-528]. Upconversion is a unique feature of certain lanthanide-based materials (with exception of a few transition metals) capable of converting infrared to visible light via sequential non-coincident absorption of two infrared photons with efficiency greatly enhanced compared to simultaneous two-photon absorption. The upconversion mechanism is based on either one type of lanthanide ion or two different lanthanide ions in proximity. The lanthanide dope ions have long-lifetime excited states, which operate as metastable states excited from a ground state to be excited again to an emission state, or transfer energy to another lanthanide ion. The lanthanide-based upconversion can provide extreme detectability, as the observed photoluminescence background is equivalent to that achieved in luminescence counting limited only by the dark current and sensitivity of the detector.
Up-converting chelates have been described in U.S. Pat. No. 5,891,656, Xiao, X. et al. (2002) Opt Lett. 30: 1674-1676; and Faris G W and Hryndza M, Proc SPIE—Int Soc Opt Eng 2002; 4626: 449-452. In an up-converting lanthanide chelate a single rare earth ion [e.g. Er(III), Tm(III) or Ho(III)] or a combination of different lanthanide ions is chelated to a mono or multinuclear complexing ligand or multiple ligands [WO 2004/086049 and Soukka, T. et al. (2008) Annals of the New York Academy of Sciences 1130: 188-200]. The ligand may or may not contain a light harvesting structure. The light collection efficiency of individual ions and chelated ligands without light harvesting structure is poor and requires relatively high excitation light intensity. Therefore, up-converting rare earth chelates can be designed to contain a ligand with light-harvesting organic or inorganic structures, e.g. another ion such as Yb(III), incorporated. The collected energies of two or more photons are transferred one after another by intramolecular non-radiative processes from the singlet to the triplet state of the organic structure, then from the triplet state sequentially to the emissive level of the rare earth ion, which then emits a single photon of characteristic emission.
Homogeneous fluorescence-based nucleic acid hybridization assays are typically based on either a quenched probe (donor and quencher in a cleavable oligonucleotide probe) [U.S. Pat. No. 5,538,848] or two energy-transfer probes (separate donor and acceptor labelled probes, which hybridize next to each other to adjacent positions). FIG. 1 describes a energy-transfer probe based hybridization assay, where two oligonucleotide probes (1 and 2), labelled with donor and acceptor fluorophores (4 and 5, respectively) hybridize (6) to adjacent positions on a complementary target sequence (3). The acceptor is excited at one wavelength (λ1) and the (energy-transfer excited) sensitized acceptor emission (8), which is dependent on the hybridization (7), is detected at another wavelength (λ2). Although, fluorescence resonance energy-transfer (FRET) is an extremely versatile technology, especially the energy-transfer probe-based assay is limited by energy-transfer efficiency (relatively low signal) and background through reabsorption of the donor emission (limited dynamic range). Further, the quenched probe based assay requires specific labeling with two different dyes and is dependent on the specificity of only a single hybridization event.
Different methods for real-time monitoring of nucleic acid amplification are presented by Koch, W. H (2004) Nature Reviews Drug Discovery 3: 749-761. For example, a FRET-pair using one dye coupled to a primer and another to an adjacently hybridizing probe has been presented by Lay, M. J. et al. (1997) Clinical Chemistry 43: 2262-2267; FRET-pair using two differently labelled adjacently hybridizing probes by Bernard, P. S., et al. (1998) American Journal of Pathology 153: 1055-1061; and competitive hybridization of FRET-pair labelled with complementary probes by Kiviniemi, et al. (2005) Clinical Biochemistry 38: 1015-1022.
Feasibility of lanthanide-label technology in fluorescence quenching based assays has been described [Karvinen J et al. (2002) J Biomol Screen 7:223-231; Karvinen, J et al. (2004) Anal Chem. 76:1429-36; Karvinen, J et al. (2004) Anal Biochem. 325: 317-25].
Two approaches have been presented for hybridization dependent formation of fluorescent lanthanide complexes. The first approach was based on a pair of oligonucleotides forming a fluorescent terbium(III) complex upon hybridization; one oligonucleotide was labeled with DTPA-terbium(III) (non-fluorescent terbium chelate) and the other with energy-donor salicylate (light harvesting ligand) [Oser A and Valet G (1990) Angew Chem Int Ed Engl 29: 1167-1169]. The second approach was based on similar formation of a fluorescent europium(III) complex but required hybridization of only one probe; the oligonucleotide probe was labeled with EDTA-terbium(III) (non-fluorescent europium chelate) and an energy-donor compound was coupled to an intercalating agent capable of binding to double stranded DNA [Coates et al. (1994) J. Chem. Soc., Chem. Commun. 2311-2312; Mullins S T et al. (1996) J Chem Soc, Perkin Trans 1 1991: 85-81; Coates J et al. J Chem Soc, Chem Commun 1995: 2311-2312; and WO 95/08642]. The first approach has also been used later [Wang et al. (2001) Analytical Biochemistry 299, 169-172; Yuan and Wang (2005) Journal of Fluorescence Vol. 15, No. 4, July, 559-568; Kitamura Y. et al. (2008) Journal of Inorganic Biochemistry Vol 102, No. 10, 1921-1931; and Kitamura, Y. et al. (2006) Nucleic Acids Symposium Series, No. 50, 105-106].
Lanthanide complex-based sensor probes have been described for detection of metal ions e.g. by Leonard, J. P. and Gunnlaugsson, T. (2005) Journal of Fluorescence, 15:585-595 and Viquier and Hulme (2006) Biology, J. Am. Chem. Soc. 128: 11370-11371. For metal cations these sensors work in a competitive manner and utilizing an antenna effect, where the binding of the antenna ligand to the lanthanide ion is blocked by another metal ion present in solution.
Quantitative 5′-nuclease based polymerase chain reaction assay (TaqMan; Applied Biosystems, Foster City, Calif.) is a nucleic acid sequence detection method wherein a single-stranded self-quenching oligonucleotide probe, containing both a fluorescent moiety and a quencher moiety, is cleaved by the nuclease action of nucleic acid polymerase upon hybridisation during nucleic acid amplification [Lie Y S, Petropoulos C J. (1998) Curr Opin Biotechnol. 9: 43-48; and Orlando C et al. (1998) Clin Chem Lab Med. 36: 255-269].
Molecular beacons are single-stranded oligonucleotide hybridization probes that form a stem-and-loop structure [Tan W et al. (2004) Curr Opin Chem Biol.; 8: 547-553; and Tan W et al. (2000) Chemistry; 6: 1107-1111]. The loop contains a nucleic acid probe sequence that is complementary to a target sequence, and the stem is formed by annealing of complementary arm sequences that are located on either side of the probe sequence. A fluorescent moiety is covalently linked to the end of one arm and a quencher is covalently linked to the end of the other arm. Due to the proximity of a fluorescent moiety and a quencher moiety molecular beacons do not fluoresce when they are free in solution. However, when they hybridize to a complementary nucleic acid strand containing a target sequence they undergo a conformational change increasing the distance between fluorescent moiety and the quencher moiety that enables the probe to fluoresce. In the absence of a complementary target sequence, the beacon probe remains closed and there is no fluorescence due to intramolecular quenching.
Both self-quenched fluorescent probes and molecular beacons are also used to monitor nucleic acid amplification processes in a thermal cycler; for example in a quantitative polymerase chain reaction the amount of fluorescence at any given cycle, or following cycling, depends on the amount of specific product. The probes bind to the amplified target following each cycle of amplification and the resulting signal upon hybridisation, and in case of Taqman probes upon cleavage, is proportional to the amount of the amplified oligonucleotide sequence. Fluorescence is measured during each annealing step when the molecular beacon is bound to its complementary target or after an elongation step when the Taqman probe is cleaved. The information is then used during quantitative PCR or quantitative RT-PCR (reverse transcriptase PCR) experiments to quantify initial copy number of amplified target nucleic acid sequence based on the threshold cycle number. For endpoint analysis, PCR or RT-PCR reactions containing molecular beacons can be run on any 96-well thermal cycler and then read in a fluorescence reader.
Sensitive and specific proximity probe-based analysis of proteins and potential in medical diagnostics has been described by Gustafsdottir, S. M. (2005) Anal Biochem 345: 2-9 utilizing proximity ligation of two oligonucleotide probes.